Nanoparticulate assisted nanoscale molecular imaging by mass spectrometry

ABSTRACT

Methods and devices for mass spectrometry are described, specifically the use of nanoparticulate implantation as a matrix for secondary ion and more generally secondary particles. A photon beam source or a nanoparticulate beam source can be used a desorption source or a primary ion/primary particle source.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/571,962 to J. Albert Schultz et al. filed on Sep. 16, 2019 andentitled “Nanoparticulate Assisted Nanoscale Molecular Imaging by MassSpectrometry”, which is a continuation of U.S. patent application Ser.No. 15/485,003 to J. Albert Schultz et al. filed on Apr. 11, 2017 andentitled “Nanoparticulate Assisted Nanoscale Molecular Imaging by MassSpectrometry”, now issued as U.S. Pat. No. 10,446,383 granted Oct. 15,2019, which is a continuation of U.S. patent application Ser. No.15/061,680 to J. Albert Schultz et al. filed on Mar. 4, 2016 andentitled “Nanoparticulate Assisted Nanoscale Molecular Imaging by MassSpectrometry”, which is a continuation of U.S. patent application Ser.No. 14/092,732 to J. Albert Schultz et al. filed on Nov. 27, 2013 andentitled “Nanoparticulate Assisted Nanoscale Molecular Imaging by MassSpectrometery”, now issued as U.S. Pat. No. 9,297,761 granted Mar. 29,2016, which is a continuation of U.S. patent application Ser. No.13/156,111 to J. Albert Schultz et al. filed on Jun. 8, 2011 andentitled “Nanoparticulate Assisted Nanoscale Molecular Imaging by MassSpectrometery”, now issued as U.S. Pat. No. 8,614,416 granted Dec. 24,2013, which claims priority to U.S. Provisional Patent Application No.61/352,678, filed on Jun. 8, 2010, all of which are incorporated hereinby reference in their entirety.

TECHNICAL FIELD

This invention relates to methods and devices for mass spectrometry,specifically the use of nanoparticulate implantation for use as a matrixfor secondary ion and more generally secondary particles. A photon beamsource or a nanoparticulate beam source can be used as a desorptionsource or a primary ion/primary particle source.

BACKGROUND OF THE INVENTION

It is well known in the literature that noble gas ions having energiesin the kilo-electronvolt (keV) range, especially neon (Ne) and helium(He) ions, cannot be successfully used as primary ions for SIMS analysisof a molecular surface. Benninghoven found that when noble gas primaryions were used to perform SIMS analysis of pure amino acids, silver (Ag)surfaces, of all the many metallic and insulating substratesinvestigated, provided the best production of small intact molecular andfragment ions. However, the use of keV cluster ions (SF₆ ⁺, Au³⁺) asprimary particles in SIMS, and ultimately, the emergence of the MALDI(matrix-assisted laser desorption) technique, eclipsed the use ofmonoatomic primary ion SIMS molecular surface analysis. Typicalsecondary ions desorbed in noble gas bombardment are either elementalions or are very weak molecular ion signals from very small molecularions (e.g. C₂H₃ ⁺). Thus, the molecular analysis of a surface by He, Neor even larger monoatomic ion or neutral atom bombardment has beenlargely abandoned for the last twenty five years.

Two critical technical issues limit the scientific community's abilityto identify biomolecular interactions that underlie cellular functionand pathophysiology. The first limitation relates to the fact that mostanalytical methods cannot detect and quantify a broad spectrum ofbiomolecules simultaneously. Current mass detection methodologies,including mass spectrometry, provide a narrow window into a smallfraction of the biomolecular universe of proteins, lipids andcarbohydrates. However, our very recent work has shown that thecombination of MALDI-Ion Mobility-orthogonal time of flight MassSpectrometry (MALDI-IM-oTOFMS) and laser post-ionization (POST) permitanalysis of both charged and neutral proteins and lipids. Thiscombination of technologies has the potential to expand the speciesdetection capabilities at least several hundred-fold for lipids,peptides, and glycoforms. The second limitation relates to the fact thatpresent-day MALDI imaging has a relatively poor spatial/volumeresolution of more than 20,000 cubic microns (1000 μm² laser spot into a20 μm thick matrix layer)—mostly because the necessary matrix layer isthicker than the tissue to be analyzed. Effective monolayer scalematrices must be found. To this end we have recently demonstrated boththe spatial resolution and sensitivity necessary for subcellularanalysis by depositing a submonolayer of aerosolized goldnanoparticulate (Au NP) matrix on the tissue surface or by implanting asubmonolayer of (1 nm) Au₄₀₀ ⁴⁺ into a 10 nm region below the tissuesurface. Both methods of Au NP deposition result in a matrix volume ofless than 9 cubic microns under the 30 micron diameter pixel (laserspot). 10 μm³ is approximately 1/100 of the volume of a 30 μm diametercell. Protein and lipid profiles and lipid imaging were measured in bothcases. Data was obtained from two sagital sections of unperfused frozenbrain tissue. A DHB (dihydroxy benzoic acid) matrix solution dropletpreferentially extracts water soluble blood proteins from the tissuewhich then dominate the MALDI spectrum. In contrast, no major bloodproteins are seen from the Au NP-implanted tissue section; instead onlyhistone and other higher mass proteins are detected. Therefore, laserimaging technologies based on Au NP implantation should be capable ofachieving subcellular molecular profiling especially when coupled withpost-ionization of desorbed neutrals in an ion mobility-oTOFMSspectrometer.

The implantation of cellular level mass spectrometry-based molecularphenotyping represents a transformational development in biomedicalscience and clinical pathology. Simple approaches, such as overlays withstandard or confocal light microscopic images can change limited andslow histochemical and immunohistochemical approaches into streamlined,broad molecular phenotyping of even small or limited biopsy samples.Similarly, it will enable quantitative analysis of individualdifferences between cells within a tissue from animals or humansubjects, such that variations between nominally similar cells can bestudied and variations in populations characterized. It will effectivelyopen a new universe of cellular proteomic and lipidomic phenotyping torapid and sensitive quantitative analysis. Laser capture microdissection followed by MALDI-MS very powerfully profiles molecules from agroup of localized cells; the MALD-POST-IM-oTOFMS biomolecularmicroscope could profile each individual cell within the group. Thiswould open a new era for routine intra-cellular biochemical profiling ofa cell populations in localized tissue regions for basic research,pathological analysis, and ultimately, clinical applications. What isneeded in the art is increased molecular detection sensitivities forsmall volumes, such a single mammalian cell.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods and devices for massspectrometry, specifically the use of nanoparticulate implantation as amatrix for secondary ions and more generally secondary particles. Aphoton beam source or a nanoparticulate beam source can be used adesorption source or a primary ion/primary particle source.

In one aspect of the invention there is an analytical instrument for thecharacterization and analysis of a sample comprising: a sample stage forpositioning a sample; a nanoparticulate beam source positioned todeliver a nanoparticulate beam to a sample on the sample stage; ananofocused primary particle beam source or a nanofocused photon beamsource, or both, the beam source positioned to deliver a beam to thesample; and, an analyzer positioned to analyze material or photonsemitted from the sample.

In one embodiment, the sample stage is an XY sample stage. In anotherembodiment, the instrument comprises a component selected from the groupconsisting of a cluster beam source, a vapor deposition system, a laserablation system, an electrospray ionization source, a molecular beamsource, an atomic layer epitaxy source, an ion beam deposition source, aKnudsen effusion cell, a magnetron sputter source, an electron beamevaporator source, an atomic hydrogen, oxygen or nitrogen source, anozonolysis source, a plasma etching source, an aerosol generator source,and any combination thereof, the component being position to delivermaterial to the sample, to the nanoparticulate beam or to both.

In one embodiment, the analyzer comprises a mass spectrometer. In aspecific embodiment where the analyzer comprises a mass spectrometer,the mass spectrometer is a time-of-flight mass spectrometer.

In one embodiment, the analyzer comprises a fluorescence spectrometer.

In one embodiment, the nanofocused primary particle beam sourcecomprises a nanofocused neon ion particle beam source. In oneembodiment, the nanofocused photon beam source comprises a nanofocusedplasmonic photon source. In one embodiment, the nanoparticulate beamsource is a nanofocused nanoparticulate beam source. In one embodiment,the nanoparticulate beam source is a nanoparticulate silver ion beamsource. In one embodiment, the nanoparticulate beam source is ananoparticulate aluminum ion beam source. In one embodiment, thenanoparticulate beam source is a nanoparticulate coreshell beam source.In one embodiment wherein the nanoparticulate beam source is ananoparticulate coreshell beam source, the nanoparticulate coreshellbeam source is a nanoparticulate aluminum/silver coreshell beam source.The instrument of claim 1, wherein the nanoparticulate beam source is analuminum nanoparticulate coreshell beam source. In one embodiment,instrument is configured as a cluster tool, comprising 1) a discreteimplantation cluster comprising the nanoparticulate beam source, and 2)a discrete desorption/analysis cluster comprising the nanofocusedprimary particle beam source or nanofocused photon beam source, or both,and the analyzer. In a specific embodiment wherein the instrument isconfigured as a cluster tool, the instrument further comprising a sampletransfer mechanism coupling the implantation cluster with thedesorption/analysis cluster.

In another aspect of the invention there is a method for the collectionof analytical data from a sample, comprising the steps of: adding matrixto the sample with a nanoparticulate beam source; thereafter desorbingchemical species from the sample using a primary particle beam source ora nanofocused photon beam source, or both; and, analyzing at least aportion of the desorbed chemical species.

In one embodiment, the primary particle beam source is a nanofocusedprimary particle beam source. In one embodiment, the primary particlebeam source is a microfocused particle beam source. In one embodiment ofthe method, the method further comprises the step of adding material tothe sample using comprising a component selected from the groupconsisting of a cluster beam source, a vapor deposition system, a laserablation system, an electrospray ionization source, a molecular beamsource, an atomic layer epitaxy source, an ion beam deposition source, aKnudsen effusion cell, a magnetron sputter source, an electron beamevaporator source, an atomic hydrogen, oxygen or nitrogen source, anozonolysis source, a plasma etching source, an aerosol generator source,and any combination thereof, the component being positioned to delivermaterial to the sample, to the nanoparticulate beam or to both.

In one mbodiment, the step of adding matrix to the sample with ananoparticulate beam source comprises adding nanoparticulate silver ionsto the sample with a silver ion beam source. In one embodiment, the stepof adding matrix to the sample with a nanoparticulate beam sourcecomprises adding nanoparticulate aluminum ions to the sample with analuminum ion beam source. In one embodiment, the step of desorbingchemical species from the sample using a primary particle beam sourcecomprises desorbing with a nanofocused neon ion particle beam source. Inone embodiment, the step of desorbing chemical species from the sampleusing a nanofocused photon beam source comprises using a nanofocusedlaser. In one embodiment, the step of analyzing comprises analyzing witha mass spectrometer. In a specific embodiment wherein the methodcomprises analyzing with a mass spectrometer, the mass spectrometer is atime-of-flight mass spectrometer.

In one embodiment of the method, the nanoparticulate beam source is ananofocused nanoparticulate beam source. In one embodiment of themethod, the nanoparticulate beam source is a microfocusednanoparticulate beam source.

In one embodiment of the method, the primary particle beam source is ananoparticulate beam source.

In one embodiment of the method the nanoparticulatebeam source is acoreshell structure nanoparticulate beam source. In one embodiment ofthe method wherein the nanoparticulate beam source is a coreshellstructure nanoparticulate beam source, the coreshell structurenanoparticulate beam source is a aluminum silver coreshell structurenanoparticulate beam source.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 illustrates a helium ion image of a silicon substrate onto whicha 2 keV silver nanoparticulate beam has been impinged;

FIG. 2 illustrates comparative SIMS data for a tri-peptide film with andwithout Ag NP in the near surface region;

FIG. 3 is a schematic diagram of a nanoparticulate beam source; and,

FIG. 4 is a schematic diagram of a coreshell NP structure.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a” and “an” means one or more than one unless otherwisestated.

As used herein, the term “soft landed” means any ion or particle whichis accelerated onto the surface with a very low energy which minimizesor completely avoids any damage to the crystallography or molecularstructure of that surface.

As used herein, “IM-MS” means ion mobility mass spectrometry, which is atechnique where an ion mobility spectrometer is fluidly coupled to anymass spectrometer

As used herein, the term “post-ionization” means the technique ofconverting uncharged atoms or molecules, liberated from a surface in aSIMS or MALDI step, into ions for analysis by the application of anionization techniques within a volume just above the surface of thesample into which the uncharged atoms or molecules have been desorbed.

As used herein, a sample denotes any material for analysis.

As used herein, the term “near surface” or “near surface region”, whenused in the context of a sample, is a thin top layer starting from thesurface gas/surface interface layer and having typical thicknesses ofabout 100 nm or less which, for example, thus encompass the firstseveral monolayers of a molecular solid.

As used herein, the term “MALDI” means matrix assisted laser desorptionionization as commonly known in the art.

As used herein, the term “MS” means mass spectrometry as commonly knownin the art.

As used herein, the term “SIMS” means secondary ion mass spectrometry ascommonly known in the art.

As used herein, NP-SIMS means secondary ion mass spectrometry which isassisted by implanting a NP into a sample to be analyzed for use asmatrix to enhance the emission of secondary ions (and in particularmolecular secondary ions) when a primary ion (which may also be a NP) issubsequently used to image the surface of the implanted sample.

As used herein, the term “TOF” means “time-of-flight” and is shorthandfor a time-of-flight mass spectrometer.

As used herein, the term “oTOF” means a time-of-flight mass spectrometerhaving a flight tube arranged orthogonally to the separation axis of apreceding separation technique.

As used herein, “MALDI-IM-oTOF” means an instruments and methods forobtaining mobility resolved mass spectra of MALDI desorbed molecular andelemental ions.

As used herein, the term “SIMS-IM-oTOF” means an instrument and methodfor obtaining mobility resolved mass spectra of secondary desorbedmolecular and elemental ions which are created during the bombardment ofa solid by an energetic primary ion beam which impinges a surface.

As used herein, the term “LMIS” means an ion source extracted fromliquid metal and can be micro or nanofocused onto a sample surface.

As used herein, the term “NP” refers to nanoparticulates, which arediscrete aggregates comprising pure atoms, alloys, coreshell structures,molecular compounds or combinations thereof where the major dimension ofthe discrete aggregate is less than a micron and often of dimensionsless than about 100 nm.

As used herein the term “nanoparticulate beam source” (or theabbreviation “NBS”) means a device capable of producing on demand a fluxof neutral or charged NPs which may be directed to a sample.

As used herein, the term “microfocused” when referring to a beam, meansthe focusing of the beam to an area of less than a mm and to about justgreater than 1 micron.

As used herein, the term “nanofocused” when referring to a beam, meansthe focusing of the beam to an area of less than a 1 micron (μm) and toabout just greater than 1 nm.

As used herein, the term “particle beam source” means a device whichdirects an energetic particle onto a surface.

As used herein, the term “microfocused particle beam source” means adevice which directs an energetic particle onto a surface within an areaof less than 1 mm and more than 1 micron.

As used herein, the term “nanofocused particle beam source” means adevice which directs an energetic particle onto a surface within an areaof less than a 1 micron and more than 1 nm.

As used herein, primary particle beam is a particle beam source whichmay be used for SIMS. This definition includes both primary ion beamsand primary neutrals beams.

Reference is made herein to the terms “primary ions” and “primaryparticles”. The meaning of primary ions should be understood to be thecustomary meaning in the field of secondary ion mass spectrometry.Additionally, the term primary particle also should be understood in thesame context (i.e., that being of a particle, that upon impingement witha sample material, gives rise to a secondary particle). In the usualcase, SIMS techniques use primary ions owing to ease of focusing ofcharged species using focusing electric fields, etc. However, in somecases, it is advantageous to use a neutral particle as a primaryparticle to impinge a sample and create secondary ions or (secondaryneutral species with can be post-ionized to form ions). Thus, it shouldbe understood that the terms “primary particles” and “secondaryparticles” are analogous to and correspond with “primary ions” and“secondary ions”, respectively, as the latter two terms are customarilyused in the SIMS literature. It merely expands the customary terms toinclude the use of neutral species in addition to ions. In this way,“ions” are a subset of “particles”. At various points herein, the terms“primary particles”, “secondary particles”, “primary ions” and“secondary ions” are used, but it should be understood that they may beinterchangeable, unless it is clear, either expressly or from thecontext, that the narrower case of ions is required rather than thebroader case of particles.

The use, described herein, of nanoparticulates (NP) as a matrix for SIMSrevives the use of monoatomic primary ions for molecular surfacesanalysis and ultimately for molecular surface imaging. FIG. 1 shows ahelium ion image of a silicon substrate onto which a 2 keV silvernanoparticulate (Ag NP) beam has been impinged using the novelnanoparticulate beam source discussed herein. The average particle sizedistribution is seen to be around 4 nm with some larger particles andother particles of less than 1 nm. Notably, the NPs can be locatedwithin the near surface region of a sample by a specially designed NPparticle beam source either by directly accelerating the particles intoa polymer surface or by first landing the NPs onto a substrate (theresult of which is illustrated in FIG. 1), and thereafter adding a pureliquid analyte or analyte solution to obtain a film in which the NP aredispersed. The size and surface volume dispersion of the NP within thesample and the near surface region of the sample now becomes theultimate determinant of the obtainable spatial resolution by microprobeor microscope based SIMS molecular mass spectrometry and other photonbased imaging analysis.

Reference is now made to FIG. 2, which shows a comparison SIMS datataken from a tri-peptide film (tri-arginine peptide (RIM)) with andwithout Ag NP in the near surface region. A NPS source was used to softland Ag NP ions of average size 4 nm onto a silicon surface with acoverage of around 20% as seen in FIG. 1. A solution of peptide wasdeposited on the Ag NP-treated surface and the same solution wasdeposited onto both a blank silicon and a stainless steel surface tocreate a control sample of RRR without Ag NP. A control film of RRR wasproduced by depositing the same solution of tri-arginine onto eithersilicon or onto stainless steel and as can be seen, the neon bombardmentproduced no significant molecular ions above m/z 100. No variation ofprimary ion energy or fluence gave any molecular ion production from thecontrol surface; all the secondary ions from the control surface areprimarily carbon and silicon elemental and small molecular ions. Bycontrast, under the same bombardment conditions, the Ag NP-treatedsurface liberated intact RRR, Ag adducted RRR, and RR fragments. Thesame results could be achieved if the RRR was first added to the polymerand the Ag NP were energetically implanted into the surface.

The exemplary data was acquired using a macro-focused (100 μm diameter)3.5 keV Ne ion beam which impinges the Ag NP surfaces onto which wasdeposited a 10 nmole/cm² tri-arginine peptide solution (approximately 30nm thick). As seen in FIG. 2, a control surface produced by depositingthe same coverage of tri-arginine solution onto either silicon or ontostainless steel yielded no significant molecular ions above 100 m/z whenbombarded with Ne under the same conditions. No variation of energy orfluence gave any better molecular ion production than what is seen fromthe control spectrum. In contrast, under the same bombardmentconditions, the RRR film containing Ag NP produced peaks at m/z largerthan 100 including Ag⁺ (at 107, 109), intact tri-arginine (488 amu), orstructural fragments RR (loss of one intact arginine at 295). The othermultiplet peaks in this mass region can also be assigned by known wateror amide loss or adduction of Na either to the intact RRR or the RRthemselves or to their structural fragments. The higher mass molecularpeaks in the 700 range are assigned to one or two Ag atoms adducted tothe RRR. Despite the measured coverage of only around 20% Ag NP byhelium ion microscopy (FIG. 1), the RRR SIMS molecular ions nonethelesscomprise nearly 40% of the ion intensity of the entire spectrum. Theremaining ion intensity is mostly concentrated in the Ag peak (107, 109)and less so the silicon peak at 28. The peaks comprising arginine (R)fragments in the range 50-80 m/z are a minority of the total ionintensity in the spectrum. The result is data which is rich in molecularinformation.

Moreover, the FIG. 2 data were obtained from a convenient, but verysmall and non-optimal, time of flight mass spectrometer (havingrelatively poor sensitivity to masses above 100) whose only function wasto demonstrate the efficient production of molecular ions by combiningNP implantation and Ne SIMS. In this so-employed primitive TOFMS, thesecondary ions are extracted from the surface into a simple linearorthogonal time of flight with a flight path of 2 inches (50 mm) and a40 mm diameter multichannel plate ion detector positioned at the firsttime focus. This MS has poor mass resolution of 100 at 100 amu andextremely poor detection efficiency for the high mass ions. The highvoltage bias on the detector face controls the time focus and impactenergy of the secondary ions onto the detector and was optimallydetermined to be 800 eV: for a molecule of mass 500, the ion detectionefficiency is only a few percent and thus the high mass molecularsecondary ions are not efficiently detected in the FIG. 2 data. If themeasured ion intensities are corrected for this well-known dependence ofdetector efficiency as a function of velocity, then the predominance inthe spectrum of the RRR and its Ag adducts are enhanced even further. Itis surprising that although the Ag NP coverage is 20% and thus the Neshould on average strike (and destroy) the tri-arginine located betweenthe NPs, molecular ions are the predominant secondary ions from thissample. The presumption is that if the Ne beam misses a Ag NP, then thebeam does strike and destroy molecules (and we do measure some smallelements and fragments comprising a low mass spectrum similar to thatfrom the control). Thus, if techniques can be used which locate theposition of the Ag NP so that the primary ion beam only hits the NP andnot the molecular film, then we would expect to even further maximizethe molecular ion intensity. If techniques for quickly locating andbombarding the NP exclusively (and thus avoiding the damage to themolecular analyte) are used, then the first and necessary step of a newapproach to image the type and location of molecules within the surfaceby NP matrix SIMS would be achieved. Alternatively, damage to themolecular analyte can be minimized by scanning the nanoprobe particlebeam or the sample (or both) in a traditional fashion while the analysisof secondary particles is resolved on a pixel by pixel basis, but whensignificant secondary molecular signal or NP signal is encountered, thescanning is halted at that spot to maximize molecular information.Re-application of NP after raster scanning can allow depth profiling ofthe near surface region.

While not intending to be bound by theory, and while the exact mechanismis not known, it is believed that a number of well understood physicalphenomena contribute to these remarkable and unexpected molecular SIMSwhich results from Ne impinging Ag NP treated biopolymer samples of FIG.2. Within a solid Ag surface bombarded at normal incidence by 10 keV Ne,the implanted Neon depth distribution is centered around a depth of 5nm. By analogy if we focus a 0.5 nm spot of 10 keV Neon onto one Ag NPof 10 nm diameter (and ignore that the deposition range of Ne may belarger within a Ag NP compared to solid silver) then Ne will transfermost of its energy either through nuclear or electronic stoppingprocesses to the Ag atoms and the Ne will remain trapped within the NP.The collision sequence of each subsequent Ne particle will cause higherand higher Ag and Ag⁺ evaporation rates. Moreover vacancies will formwithin the remaining portion of the NP where trapped residual Ne gaspressure can accumulate and exert large internal forces which furtherweakens the intennetallic binding of the Ag in the NP and assists thevaporization of the remaining portions of the partially sputtered NP.This is in analogy to surface implantation of keV noble gas ions intometals where such phenomena are well known. Moreover, NP plasmoniceffects can increase a photon absorption cross-section of the NP at aplasmon resonance which can interconvert the photon energy through threebody interactions into kinetic ejection of atoms or ions. If the Neprimary ion excites this plasmon resonance, then this may be yet anotherway to increases the coupling of inelastic energy loss of the primaryNeon ion energy into the Ag NP.

Molecular SIMS with a 20 nm Satial Resolution by Combining 10 nmNanoParticulate Deposition with 0.5 nm He or Ne or Other Nanofocused Ionor Photon Irradation

One preferred embodiment is to use the recently developed, commerciallyavailable He or Ne ion nanoprobes which can attain spatial focuses downto 0.25 nm. An experimental combination which would then define theultimate spatial resolution of a molecular SIMS ion microprobe is asfollows. In a nanoparticulate beam source (NBS), a NP ion is created andoptimized for size, shape, and elemental or molecular composition and iseither soft landed from the NBS onto or implanted into the surface ofinterest. The NBS and the surface may be in a separate chamber, or theseparate chamber may contain a vacuum lock between the NBS and the ionmicroprobe or the NBS may be contained within or fluidly coupled withthe ion microprobe itself. The partial coverage of NP actually givesfairly uniform spacing of the nanoparticulates on a variety of surfacesby a self-avoidance mechanism and this persists into the near surfaceregion. One way to achieve nearly uniform dispersion of NP throughoutthe near surface region, is to use a sequence of first high energy(e.g., 10 keV) implantation of a known dosage of NP ions from the NBS,followed by a second dosage with an 8 keV NP energy so that evershallower depths within the near surface region receive uniformlydispersed NP comprising around 20-30% of the volume. Further steps ofreducing the NBS energy and dosing will sequentially fill in theremaining unimplanted volume of the near surface region until at thelast a soft landing dose can be applied to the surface of the sample. Atthis point in the sequence, the sample will have a nearly uniformvolumetric dispersion of the NP between the molecules comprising thenear surface region. In practice, we have found by sputter depthprofiling that the NP-implanted near surface region of a biologicaltissue can be uniformly implanted with NP throughout a 50-100 nm depthdepending on the energy and size of the NP and the type of tissue.

Thus, the smallest pixel dimensions of any microprobe analysis based oninteracting with a single Ag NP is the diameter of the NP itself, whilethe pitch (the unbombarded area) is the average distance betweenparticles. For the Ag NP on silicon shown in FIG. 1, this becomes a 4 nmpixel size and about a 15 nm pitch. We now introduce a combinedmicroprobe, combining the use of both He and Ne ions. Helium ions arefar less damaging to the first 200 nm of a biological tissue than Ne forthe same dose and kinetic energy. Thus it is possible, using the leastdestructive dose and energy He ion microprobe, to first ion etchfiduciary marks on the sample surface near the sample area of interestand then to quickly obtain a pixelated secondary electron contrast imageof the position (relative to these fiduciary mark) of each Ag NP on thesurface of the tissue. Once a map has been made of the location of theNP, the helium gas can be switched to Neon and by relocating the Heliumion etched fiduciaries marks, the Neon can then follow the helium ionenergy map to sequentially bombard only the areas containing each of theAg NP. One type of analysis of the material which can be very potent isof course the NP-assisted molecular SIMS. The secondary ions aregenerated after one or more neon gas ions strike the NP. The MS of thedirect ions and any post-ionized analytes (if a laser is used to ionizeejected secondary neutrals) are then measured in an analyzer; preferablya mass spectrometer, and more preferably an IM-oTOFMS. Once the Ag NPmatrix has been destroyed at each location, no more useful molecularinformation can be obtain and the Neon beam is ideally moved to the nextAg NP location. In this way a map of Neon ion-induced secondarymolecular ion emission can be overlayed with the Helium ion map of theAg NP locations.

Other nanofocused ion beam sources (or nanofocused photon beam sourcesfrom nanophotonic light or synchrotron radiation sources) can initiatethe desirable serial evaporation of a single NP. These include, e.g.,Ga, In, or Au from a LMIS. Since the LMIS focal spots are in the rangeof 20-50 nm, their use for NP-SIMS imaging requires the implantation ofcomparable diameter NPs in the range of 20-50 nm. On the other hand,nanofocused Ga and In are less than ideal for SIMS because of the quickmetallic implant contamination of the impact zone which not onlycomplicates SIMS spectral assignments but also quickly reduces oreliminates the sputter ion yield of most elements and molecules fromthese nanoregions as the analyzed area metalizes. However, there areother gas sources such as duoplasmatrons which can produce nanofocusedbeams with nearly similar focal properties as the LMIS but from avariety of gas sources including: all of the noble gases; especiallyuseful are high currents of double and triple charged Kr and Xe, as wellas nitrogen and oxygen. The higher energy attainable with a triplycharged Xe is useful for sputtering the larger nanoparticulates (e.g.,50 nm or greater). This is helpful because the focus of theduoplasmatron will be less than 100 nm (although a theoretically smallerfocus of 20 nm may be achieved with additional engineering). Moreover,oxygen, and nitrogen nanofocused ion beams are available from theseduoplamatron sources as well which enhance the SIMS sputter ion yield.The molecular images which could be attained from such a source would belimited by a focal spot size of 75 nm, which is now being attained usingArgon. However, the source is very versatile and relatively much lessexpensive.

The NP particle beam generated by the NBS source could itself benanofocused into a primary particle source to be alternately used as aSIMS probe after implantation. A 2 nm Au NP can create a high sputteryield of intact molecules of m/z of 1500 when used to bombard pure filmsof lipids and peptides. Moreover, this yield persists after prolongedbombardment of the surface, indicating that the damage created in thesputtering event was ejected from the sputter crater along with theintact molecular ions while the remaining biopolymer around the craterremained intact. These observations led the inventors to develop asputter profiling sequence in which a focused Au NP was rastered across,and implanted within, the near surface region below the biopolymersurface. Notably, during this raster implantation of the Au NP, aspatially resolved SIMS was acquired also. Then the implanted nearsurface region was further rastered with a focused pulsed laser so thatthe implanted Au NP, which had just yielded SIMS, was used as a matrixto liberate MALDI ions. Earlier work in this area never recognized thatthe implanted Au NP could function as a SIMS matrix in addition tofunctioning as a MALDI matrix. The use of the LMIS Au₄₀₀ ⁴⁺ NP ionsource to implant the Au NP for use as a SIMS matrix and to thenimmediately microfocus the Au₄₀₀ ⁴⁺ NP ion beam for use as a primary ionto obtain a NP-SIMS image assisted by the previously implanted Au NP wasnever considered either.

Accordingly, what we now show is that if one first uses the NP implanterto soft land or implant a sample with a spatially distributed coverageof NPs for use as a SIMS matrix then an energetic NP can also be chosento be used as the primary ion for generating additional SIMS informationfrom this NP implanted surface. The same type of NP can be used both asimplanted matrix and subsequently also as the primary ion, and thechoice of the NP type for each role may be the same or different. TheNBS allows rapid interchange between implanting one layer with one NP(e.g. Au NP) and another near surface layer with, for example Ag NP. Yeta third different NP (e.g. Al NP) might be rapidly selected and used asa nanofocused NP primary particle beam in order to obtain SIMS imagesfrom the two implanted layers by accelerating the primary NP to high keVenergies and collecting the secondary ion mass spectra as a function ofNP beam position. In this mode, the spatial resolution is that of thenanofocused NP beam (estimated at about 500 nm at best). However, it iswell known that, if the secondary electrons are properly collected andmagnified onto a position sensitive detector, then the co-incidencesbetween the secondary ions and the electrons will be correlated fromeach and every primary NP ion collision with the surface (predominantlythe collisions will be with the NPs distributed on the surface). Typicalmagnifications of up to 50 can be obtained using the secondaryelectrons, so a 500 nm micro-focus of the NP primary ions would beenhanced to a spatial resolution of well under 50 nm for this example. Arecent SIMS microscope using Au₄₀₀ ⁴⁺ NP as a primary ion has beenconstructed around such a co-incident camera detector concept; however,its sensitivity to molecular SIMS relies solely on the large molecularion sputtering yield inherent in the collision of the primary Au NP ionwith an untreated biological surface. We teach here that thismethodology can be vastly improved by using an implanted NP as matrix toenhance SIMS analysis of a molecular or elemental sample followed by theuse of a second nanofocused NP for use as the primary ion for obtaining.The NPs chosen as the matrix and as the primary ion need not be the sameand neither must necessarily be Au NP.

We have identified another factor which can affect the microscopictrajectory of any of the above listed primary ions as they near thesurface. The charge on the primary ion will induce surface dipoles inthe implanted NPs as the negatively charged primary NP (or positivelycharged Ne⁺ or Xe⁺³) nears a sample which has been prepared with closelydispersed and disposed NP implant. In the case of the negatively chargedNP, there are at least two induced dipoles one on the primary particleand one on the many partial dipoles distributed on the surface NPparticles closest to the point of ion impact. As the primary ion getsvery close to the surface the induced dipoles in the implanted NP canguide the primary ion toward the implanted nanoparticulate which isclosest to the approaching primary NP. In this way the primary NPpreferentially hits the implanted NP. This focusing effect is maximizedas the energy of the primary particle is lowered. Also in the case ofXe⁺³, the multiple charge can create multiple holes (i.e., positivecharges) within the implanted matrix NP by stripping multiple electronsfrom NP as the Xe⁺³ approaches, and just before colliding with thesurface. This imparts around 100 eV of potential energy into the matrixNP. The dipole steering of multiply charged keV-energy, monoatomic iontowards a particular implanted metallic NP, and the amount of potentialenergy dumped into that particular NP is enhanced as the chargemultiplicity on the monoatomic ion increases. Monoatomic ions withcharged states of approximately e.g., Au⁺⁶⁹ or higher which contain onthe order of hundreds of keV of potential energy can be prepared withspecial ion sources and have been used to liberate intact peptides fromsurfaces. These intact peptide ions are not desorbed by the collision ofthe multiply charged primary ion, but by the potential energy andsubsequent coulomb explosion by mutual repulsion of positively chargedsurface ions which ensues as electrons are locally removed from atomsand molecules on the surface, to fill the deep potential wells of theapproaching and nearly naked multiply charged ion.

Thus NP SIMS can be usefully attained when the primary ion is a highlycharged monoatomic ion which transfers potential and not kinetic energydirectly to a specific implanted metallic NP which subsequently explodesand carries away surrounding molecules from the surface into the gasphase. The preferential electron extraction from the implanted metallicNP is logical for two reasons: 1) the multiply charged primary ion isgoing to be closer to the surface NP than to any other molecule on thesurface and 2) it is much easier for an ion to obtain an electron fromthe energetically and spatially available electron energy bands in themetallic NP compared to the tightly bound valence electrons of acovalently bonded molecule. Thus for example if Ag NP were used as theimplanted NP SIMS matrix then multiple Ag⁺ ions would be created anddesorb violently in one concerted motion which would lift moleculeswhich were nearby. If these molecules contained double bonds or aromaticsubstituents then the Ag⁺ would form a radical cation directly withthese molecules to form analytically useful secondary ions. Nano-imagingcould be achieved by rastering a nanofocused multiply charged primaryion as well as using a position and time focusing camera detector tomeasure the time and spatial origin of secondary electrons or secondaryhydrogen ions from the surface in co-incidence with the other positiveand negative secondary ions.

The NBS Can Create and Surface-Modify Unusual NP Matrices for Gas PhaseImplantation

Newer and even more effective monolayer scale matrices must be found forpolymer and molecular analysis of complex molecular surfaces. Recentcommercial developments of NP sources based around RF magnetron providemuch flexibility in the creation and manipulation of NPs. For example,not just Au NP, but any metallic or metal alloy, can be converted intoNPs which have, for example, a 4 nm diameter with a range of clustersizes between 2 and 6 nm full width half max.

The mean diameter of these particle size distributions can be shifted,for example, between 3 and 15 nm depending on source parameter. Largerparticle sizes are possible as well. In fact, the particle size can beprecisely tuned by varying power and gas flow rate to achieve +/−10%FWHM particle size distributions centered around a mean value which canbe selected, for example, within the range of 1 nm up to 20 nm.

One embodiment of our NBS (100) is shown in FIG. 3 and combinesmagnetron based NP source (200) which produces negatively charged NP(20) which are guided through a series of differentially pumped (10)vacuum regions which house ion mobilty ion trap growth section (210), abeam cooling region (220), a beam acceleration and focusing section(230), and a sample chamber (240) which may also house all the sampleanalysis instrumentation as well. Note that multiple deposition sources(110) can be attached to each of these sections (210, 220, 230, 240) toadd material to the surface of the NP (20) as they emerge from the NPsource (200). The selection of multiple deposition source (110) in eachof the three regions may be different from region to region according tothe needs to treat the NP particle at each location. The types ofmaterial deposition sources are chosen from the group from the groupconsisting of a cluster beam source, a vapor deposition system, a laserablation system, an electrospray ionization source, a molecular beamsource, an atomic layer epitaxy source, an ion beam deposition source, aKnudsen effusion cell, a magnetron sputter source, an electron beamevaporator source, an atomic hydrogen, oxygen or nitrogen source, anozonolysis source, a plasma etching source, an aerosol generator source,and any combination thereof, with the component being positioned todeliver material to said sample, to the nanoparticulate beam or to both.

Al, Ag or Au NP implantation works very well in this NBS (100) withoutfurther addition of material. However, the capability to try variousother metal NP combinations is equally compelling. Au is relativelyinert and does not affect the molecular ions nor does it form stablemetal adduct ions (although the stable neutral Au, by adducting withother molecules, can have a profound effect on post-ionization ofneutral molecule Au complexes). Other metals behave differently such assilver which makes a strongly charged Ag adduct ion (and neutral adductsas well) to many molecules. Mixing NPs or alternating deposition of twodifferent metal NP types in between imaging raster scans can be a toolfor producing desired effects on the surface including fragmentation orionization of previously undetected molecules. The power of the NPimplanter can furthermore be augmented by co-deposition of a fewmonolayers of traditional organic acid matrix (or other desired acid orbasic additive(s)) which can chemically ionize independently of theeffect of the chemical characteristics of the NP.

The above approach is a significant alternative to liquid droplet matrixdeposition schemes which are plagued with unavoidable effects of fluidphysics which are common to any droplet deposition technique. If onewants to achieve droplet sizes approaching 25 μm, then the evaporationrate is so high that the viscosity of the solute laden droplet rapidlyincreases and rapid crystallization of the matrix occurs on a time scaletoo fast to allow solvent extraction of surface molecules into thecrystal before it is dried. Moreover, it takes several hours to depositthe matrix mixture onto the tissue even at 50 micron droplet sizes usingink jet printer technology.

An alternative is found by using the NP implanter is to decouple theaddition of NP matrix, solvent (if solvent is even necessary) and aciddeposition into three different sources. Multilayer liquid phases ofsolvents such as water may comprise only a few monolayers of fluid ontothe surface. This “solvent” thickness can be controlled by temperatureand water vapor pressure, whereas acid or base can be independentlyadded from a gas effusion source during the time that the NPs are beingsoft landed or implanted. Moreover, this procedure can be carried outsimultaneously on multiple sagital sections as previously mentioned.These different types of sources, such as Knudsen effusion cell,controlled reactive or inert gas flow, sputter deposition sources, laserdeposition sources, plasma treatment, reactive gas etching, reactive ionor metastable atom surface treatment are well known within the filmgrowth and molecular beam epitaxy community. Surprisingly, thistechnology has never been systematically applied to matrix deposition ofmixed thin films onto biological or synthetic polymer surfaces. Themolecular analysis of these hugely important classes of materials haslain dormant as a result.

Another possible combination for the sequential or simultaneousapplication of multiple thin film components using multiple cells fordeposition onto a complex molecular surface is 1) NP implantation, 2)gas adsorption (e.g., NH₃, H₂O, SO₂, NO₂, O₂, CO₂, ozone, HF, HCl, HBr,or HI, Iodine, or volatile organic acids such as acetic acid, volatileorganic bases such as ethanolamine, 3) ion or elemental addition toimprove ionization efficiency or cationization efficiency such as anyalkali or second row alkaline earth (e.g. Ca) which can be provided aspure neutrals by dispenser sources, or as ions by surface ionizationsources, 4) non volatile additive which can be applied in aerosolizedform through a droplet source, 5) volatile organometallic compounds. Thesequential or simultaneous addition of elements or molecules from any ofthese sources can be monitored and controlled through many of the knownmetrologies in thin film growth which measure the incident flux ofadditive elements or molecules toward the surface or detect surface filmgrowth and these include, but are not limited to: 1) capacitancemanometer 2) fast ion gauges, 3) crystal microbalances, 4) massspectrometry of surface or gas phase compositions, 5) light scattering,6) electron or photon spectroscopies or spectrometries includingellipsometry or other interference based film thickness measurementtechniques, 7) fast current measurement of ion beam flux toward thesample or into the sample.

Optimization of the NP composition and growth process as a function ofNP type and size is now possible. Non-limiting elements forconsideration include Li, Be, B, C, N, Na, Mg, Al, B, Ti Ag, Au, Cu, Zn,Zr, Fe, Mn, Mo, Co, V, Pt, Ni, Cr Ir, Bi, Pb, and alloys of these metalNPs. The size at which these NPs are most efficient typically variesfrom 1 nm up to 200 nm. In the case of plasmonic NPs such as Ag NP, theadditional combination of extensive Surface Enhance Raman Spectrometry(SERS) is possible which yields structural information about moleculeswhich are on or close to the Ag NP and may be possible also with Al NPand other more reactive plasmonic metals. The Raman spectrum can benon-destructively obtained after the addition of the Ag NP but prior tothe ablation of the surface into the mass spectrometer (with ions of Neor He or Ar or Xe or by another large NP or by simultaneous laserassisted ion bombardment, or by laser desorption alone). Similaradvantages from the size engineered NPs may confer to fluorescencemicroscopy as well.

Coreshell NP Can be Contructed to Maximize Optical Absorption andMinimize Destructive Heat Transfer to Analyte Molecules

FIG. 4 shows a NP (20) which is made up of a core (23) metal and a shellstructure (22). Such a structure is called a coreshell NP. The shell canoften have a third layer (21) which may be a desirable (or undesirable)oxide layer for example. In the case of AgAl NP coreshell the Ag core issurrounded by an aluminum shell, but the aluminum shell may also have athin layer (21) on its own surface which is often an oxide layer. Suchan outer oxide layer is also almost always the case for pure Al NP wherethe center is pure Al and a thin oxidation layer is present on thesurface. In some cases it is desirable to form this oxidized layer sincethe oxide is a thermal insulator. If Al NP is used as a SIMS or a MALDImatrix, the thermally insulating oxide structure holds the heat in thecenter of the particle thus preventing unwanted thermal degradation ofthe surrounding analyte. In many methods of NP formation, the oxidelayer on such active metals is unavoidable.

The NBS (100) can control a coreshell structure in an unprecedented waysince all of the vacuum regions can be made from UHV compatiblematerials which eliminate or reduce the presence of oxygen or water.Thus, if oxidation is desired then ozone or oxygen gas can be presentedto the pure gas phase NP as it exits the NP source (200); however, ifthe aim is to prevent the oxidation then this can be done as well byputting the appropriate deposition source (110) into the region (210)just as the particles are exiting the NP source or in region (220) wherethe particles are being spatially trapped.

For example, Al can form an extremely thermally non-conductive nitridelayer which also is extremely hard, is a chemically inert surface and isa wide bandgap material which is transparent to UV and VUV photons. If ahigh fluence atomic nitrogen source (110) is used in the NP source exitregion (210) a nitride layer could be formed on top of the pure Al NP.Theoretical predictions of the influence of Al NP size on its opticalabsorption show that as the size if varied from 100 nm down to 10 nm itsoptical absorbance maximum varies from a broad absorbance which ispartly reflective between 300 and 150 nm to a narrow plasmon absorbancewhich is centered at 150 nm and predicted to have very little componentof reflectivity. Thus if we created a pure Al NP of 10 nm diameter andput a thin AlN “greenhouse” shell around this pure particle before itcould oxidize then we have a AlN NP structure which could be highlyuseful in localizing heating into the interior of the Al core by thefluorine excimer wavelength of 157 nm. The 157 nm photon could getthrough the nitride layer but the ensuing heat from its absorption bythe Al core could not get out. Moreover, the tensile strength of thecoating would tolerate an increased internal pressure so that when theparticle began evaporating it would do so forcefully and quickly. Asimilar mechanism would also be anticipated by Neon (or even He)bombardment. The aluminum nitride coating is radiation hard so the fastnoble gas ion would create little damage as it penetrated the outernitride layer and then dump all its kinetic energy into the pure Alcenter and the AlN shell would locally contain the heat. Aluminumcoreshell structures, whether oxide or nitride, could auger not onlyadvances in using excimers and ions for molecular surface analysis, butmight give advantages in aspects of excimer laser surgery. Similarnitrogen, oxygen, and carbon surface chemistry of the shell or corestructure can be achieved with boron nitride, carbide coatings,titatnium nitride, titanium oxide, silicon nitride oxide and carbide. Anespecially good guide for these types of chemistry is to look to thesemiconductor industry for the processes and materials which they use tocreate metallic conductors on insulator, for techniques and materials tocreate existing and state of the art structures, transistor drain andgate features, diffusion barriers. Any materials and depositiontechnologies used by the semiconductor industry would be entirelycompatible with NBS (100) for creating similar gas phase complex NPstructures.

Additionally, when the small Al NP structures are optically excited inthe UV and VUV, good energy transfer to naturally occurring flourophoresin biological structures has been shown theoretically. The search for ametal NP plasmonic alternative to synthetic labeling of biomoleculeswith large fluorescent dye molecules in biological systems is beingactively pursued. Thus, the capability of the NBS (100) to locatespecially prepared unlabeled NP fluorescent donors into biologicalsurfaces for exciting the intrinsic fluorescence of biomolecules couldprovide dual use for this instrument. Alternatively, these NP probescould have use in confocal microscopy and fluorescence microscopy. Oneset of experiments would be to implant a NP which is designed for theseoptical techniques and is also an efficient SIMS or MALDI NP matrix.Then the non-destructive optical imaging could be performed, and thenthe molecular map determined by either nano-SIMS or nano-MALDI.

Other NP or coreshell NP structures which form strong plasmons but whichare prone to oxidation (Li, Na, Mg, Al, Si) can be protected by shellcoatings which are impervious to oxidation. If the NP implant isdesigned carefully, there can be a metallic coreshell particle with ametallic or semiconducting core and inert outer shell such as Au NP andparticularly Pt NP which is impervious to surface reactions and tointer-diffusion of elements even such as hydride ions into or out of theshell.

NP Interior Can be Loaded with Reducer or Oxidant (e.g., O or H)

Mg NP and MgNi NP should absorb extremely large amounts of hydrogeninterstitially. An atomic H source can be used to provide the H to thepure metal or metal alloy NP prior to adding a capping shell layer.Using a Pt or Au vapor produced with a magnetron or evaporation sourceto coat the NPs is now possible. Another example of such a usefulparticle might be a Si or SiH core with a Pt/PtSi shell. Silane gas isextremely useful for silicon atomic layer eptiaxy and the PlatinumSilicide is an extremely good diffusion barrier. Lithium aluminumhydride is a known reagent in organic chemistry which can only beprepared and used stably in ether could be capture in a coreshellstructure and delivered unreacted to a sample surface. Mg can similarlybe used as a Grignard reagent. Lithium Aluminum Hydride and NaBH₄ arewell known reducing agents and can be encapsulated within the core of acore shell NP. All of the Group IA and Group IIA metals such as lithiumor magnesium can be incorporated either directly onto the sample surfaceor isolated with the core of a NP coreshell structure by getter sourcesor by ion sources. Copper lithium alloys are amenable to coreshellincorporation.

Another useful feature of the NBS (100) especially in the ion mobilitytrapping region (210) or cooling region (220) would be the associationof large organic molecular ions which can either be prepared andintroduced with an aerosol generator or electrospray ionization source(110) with trapped NP. If the NPs are held long enough the production ofsubstantial numbers of twinned NPs begin to dominate the gas phase NPcomposition. Any large molecule (either charged or uncharged) canquickly find a NP twin partner in this region and the resulting NPmultimer will remained charged and can thus be implanted even within asmall region onto the sample. Non-limiting examples of these types oforganic molecules would be enzymes such as lipidases, proteases,fluorescent probes, drug molecules, any isotopically labeled biomoleculeto be applied as a calibration standard, organic matrix molecules.

The NBS (100) can be incorporated directly into analytical tools such asa fluorescence microscope, a mass spectrometer, a confocal microscope,electron microscope etc or it can be used in conjunction with any ofthese instrument via being part of a cluster tool—i.e., a collection ofinstruments which pass a sample (by vacuum interlocks and manual orautomated sample transfer devices) from one processing or analysisstation to another. A particularly powerful cluster tool for biologicaltissue preparation and imaging by multiple techniques would be one whichstarts a tissue sample from the tissue cryotome where the tissue sampleis prepared and mounted on cooled sample mount where it then transfersunder controlled atmosphere and controlled cold temperature (so that itnever warms) successively from one analytical station to the next. TheNBS and the MALDI-IM-oTOFMS would be one of such stations.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. An analytical instrument for the preparation,characterization and analysis of a sample, the instrument comprising: acooled sample stage for positioning a sample; a plurality ofnanoparticulate beam sources positioned to deliver a beam of energeticnanoparticulates into or onto the sample, wherein the plurality ofnanoparticulate beam sources comprises: a first nanoparticulate beamsource configured to generate first nanoparticulates selected to have afirst size and energy and to deposit the first nanoparticulates as amatrix on the sample, and an additional nanoparticulate beam sourceconfigured to generate, independently of the first nanoparticulate beamsource, second nanoparticulates selected to have a second size andenergy and to deposit the second nanoparticulates as coreshellnanoparticulates as part of the matrix on the sample; a first imagingbeam source configured to etch fiduciary marks on the sample and to scanthe sample to produce a secondary electron contrast image and togenerate a map of locations of nanoparticulates on or within the sample;a second imaging beam source configured to scan the sample according tothe map to desorb exclusively from a volume on the sample defined by thenanoparticulates using the fiduciary marks; and an analyzer configuredto detect at least one of particles or photons emitted from the sample.2. The analytical instrument of claim 1, wherein the second imaging beamsource comprises at least one of a nano-focused Ne, Ga, In, Au, Kr, Xe,nitrogen, or oxygen ion particle beam source, a nanofocused Aunanoparticulate or Bi nanoparticulate ion beam source, or a nanofocusedpulsed UV or VUV light source tuned to an optical plasmon resonance ofthe matrix.
 3. The analytical instrument of claim 2, wherein the firstimaging beam source comprises at least one of a nano-focused Helium, anano-focused Neon ion beam source, a nano-focused electron beam source.4. The analytical instrument of claim 1, wherein the firstnanoparticulate beam source comprises an instrumental component, whereinthe instrumental component comprises a water vapor source, wherein theinstrumental component is configured to produce a pure nanoparticulateion beam when the water vapor source is closed and configured to producean oxide nanoparticulate beam when the water vapor source is open. 5.The analytical instrument of claim 4, wherein the first nanoparticulatebeam source is configured to produce pure and oxide nanoparticulates ofat least one of Li, B, C, Al, Mg, Si, Ti, Cu, Zn, Zr, Ni, Cr, Ag, Au, orPt, or alloys thereof.
 6. The analytical instrument of claim 4, whereinthe first nanoparticulate beam source comprises at least one of asilver-gold alloy nanoparticulate beam source, a silver-magnesium alloynanoparticulate beam source, a silver-aluminum alloy nanoparticulatebeam source, a magnesium-aluminum alloy nanoparticulate beam source, acopper lithium alloy nanoparticulate beam source, a lithium-aluminumalloy nanoparticulate beam source, a gold-copper nanoparticulate beamsource, or a lithium-silver alloy nanoparticulate beam source.
 7. Theanalytical instrument of claim 4, further comprising a molecular vaporsource configured to add organic material to at least one of the purenanoparticulate ion beam or the oxide nanoparticulate beam prior tocollision with the sample or to the sample.
 8. The analytical instrumentof claim 4, further comprising a vapor source configured to addisotopically-labelled molecular standards to at least one of the purenanoparticulate ion beam or the oxide nanoparticulate beam prior tocollision with the sample or to the sample.
 9. The analytical instrumentof claim 4, further comprising a cesium source configured to add cesiumto at least one of the pure nanoparticulate ion beam or the oxidenanoparticulate beam prior to collision with the sample or to thesample, wherein the cesium source comprises at least one of a cesium ionsource or a neutral cesium vapor source.
 10. The analytical instrumentof claim 4, further comprising an iodine source configured to add iodineto at least one of the pure nanoparticulate ion beam or the oxidenanoparticulate beam prior to collision with the sample or to thesample.
 11. The analytical instrument of claim 4, further comprising ahydrogen-iodide (HI) source configured to add hydrogen-iodide to atleast one of the pure nanoparticulate beam or the oxide nanoparticulatebeam prior to collision with the sample or to the sample.
 12. Theanalytical instrument of claim 1, wherein the second nanoparticulatebeam source comprises at least one of a gold nanoparticulate beam sourceor a silver nanoparticulate beam source, the second nanoparticulate beamsource comprising an instrumental component, wherein the instrumentalcomponent comprises a water vapor source, wherein the instrumentalcomponent is configured to produce at least one of a goldnanoparticulate ion beam for second nanoparticulates or a silvernanoparticulate ion beam for second nanoparticulates when the watervapor source is closed and configured to produce a corresponding goldoxide nanoparticulate beam for second nanoparticulates or silver oxidenanoparticulate beam for second nanoparticulates when the water vaporsource is open.
 13. The analytical instrument of claim 12, furthercomprising a molecular vapor source configured to add organic materialto the second nanoparticulates prior to collision with the sample or tothe sample.
 14. The analytical instrument of claim 12, furthercomprising a vapor source configured to add isotopically-labelledmolecular standards to the second nanoparticulates prior to collisionwith the sample or to the sample.
 15. The analytical instrument of claim12, further comprising a cesium source configured to add cesium to thesecond nanoparticulates prior to collision with the sample or to thesample, wherein the cesium source comprises at least one of a cesium ionsource or a neutral cesium vapor source.
 16. The analytical instrumentof claim 12, further comprising an iodine source configured to addiodine to the second nanoparticulates prior to collision with the sampleor to the sample.
 17. The analytical instrument of claim 12, furthercomprising a hydrogen-iodide (HI) source configured to addhydrogen-iodide to the second nanoparticulates prior to collision withthe sample or to the sample.
 18. The analytical instrument of claim 1,wherein the sample comprises a biological sample.
 19. The analyticalinstrument of claim 1, further comprising a vapor source directed at thesample such that vapor is deposited onto the sample prior to, during, orafter impingement of at least one of the first or second nanoparticulatebeams.